Regulation of a variety of responses in both eukaryotic and prokaryotic cells involves families of proteins that function as molecular switches to turn on and off particular effector functions. The switch between inactive and active states results from conformational changes that are induced either by covalent modifications of protein side chains or by binding of small molecules. In prokaryotic cells, a family of response regulator proteins composed of homologous switch domains and specific effector domains, mediate responses to changing environmental conditions. The regulatory domain is turned on by phosphorylation of an aspartate side chain and turned off by hydrolysis of the acyl phosphate by an intrinsic phosphatase activity. Phosphorylation of the regulatory domain occurs via phosphotransfer from an associated histidine protein kinase. These phosphotransfer mediated signal transduction systems are widespread throughout the bacterial kingdom and regulate processes such as cell motility, differentiation, transport, metabolism, and establishment of host/pathogen interactions. The proposed research focuses on structure/function analysis of these phosphorylation-activated switch domains with the goal of understanding the molecular mechanism of their action. Specifically, what are the mechanisms of phosphotransfer and phosphate hydrolysis? What is the nature of the conformational change that is induced by phosphorylation? And how does the regulatory domain transmit its effects to the effector domain? The bacterial chemotaxis protein, CheY, which regulates flagellar rotation, is representative of the phosphorylation-activated regulatory domains. This 128 amino acid single domain protein has a classic alpha/beta fold consisting of a central five- stranded parallel beta sheet flanked on both sides by alpha helices. The active site, located at the C-terminal edge of the beta sheet is composed of residues that are highly conserved among the family of bacterial response regulators. The active site acidic pocket, formed by a cluster of carboxylate side chains, is the site of phosphorylation and divalent metal ion binding. Using CheY as a model regulatory domain, the mechanisms of phosphotransfer and dephosphorylation will be addressed by constructing site specific mutations, characterizing the phosphorylating/dephosphorylating activities of these altered proteins using small molecule phospho-donors, and determining the structures of relevant mutant proteins by X-ray crystallography. The X-ray structure of the active conformation of CheY will be approached both by screening for mutations that stabilize the active form, and by using small molecule phospho-donors capable of phosphorylating CheY in crystals. As a step towards determining the mechanism of regulation of effector function via phosphorylation of the switch domain, structural analysis of two additional response regulators with attached methylesterase (CheB) and DNA binding (OmpR) effector domains will be initiated.